GC–MS profiling and antibacterial activity of Solanum khasianum leaf and root extracts

Background Solanum khasianum is an important medicinal herb of the Solanaceae family. The present study was focused to determine the bioactive compounds in S. khasianum leaf and root extract by GC–MS analysis and their antibacterial activity by agar well diffusion method. Results Sixteen bioactive compounds were detected in leaf extract and thirty-two compounds in root methanolic extract by GC–MS. The major potent compounds identified in leaf and root extracts were heptadecane 9-hexyl (43.65%) and stigmasterol (23.18%). The root extract showed increased antibacterial activity than leaf extract. Conclusion These extracts possessed significant antibacterial activity against the tested bacterial isolates in dose-dependent manner. This study provides the phytoconstituents, antibacterial property and scientific evidence for the traditional claim and use of S. khasianum.


Background
Nature is the richest source of several natural therapeutic compounds. Solanaceae, one of the largest plant families with huge and varied secondary metabolites, used in the management of several ailments. The medicinal value of plants can be correlated to different phytochemicals, as they offer a wide diversity of pharmacological activities. Due to these pharmacological properties, a great attention has been derived toward the medicinal plants.
Solanum khasianum is a traditional medicinal plant belonging to Solanaceae family. The plant was known to possess potential alkaloids (solasodine, solasonine, solanine, solamargine and khasianine) that represent an alternative source of medicine (Kaunda and Zhang 2019;Chirumamilla et al. 2021). The berries of S. khasianum was reported to possess anticancer (Rosangkima and Jagetia 2015), antibacterial (Pavani and Shasthree 2021), anti-inflammatory ), antioxidant, anti-diabetic and anti-cholinesterase properties (Gogoi et al. 2021). Besides these, the plant is used traditionally to treat several other diseases like filaria, smallpox, whooping cough, rheumatism, trachoma, bronchitis, snake bites, skin and tooth infections .
To the best of our knowledge there is no information on the chromatographic analysis of S. khasianum leaf and root extracts. Hence, the current study was focused to determine several bioactive compounds in S. khasianum leaf and root extracts by GC-MS analysis. The antibacterial property against gram positive and gram negative bacteria isolates was also revealed by agar well diffusion method.
Page 2 of 10 Chirumamilla et al. Bulletin of the National Research Centre (2022) 46:127 The powdered plant material was mixed with methanol (1:10 w/v) and incubated at 22 °C in an orbital shaker at 120 rpm for 48 h. The samples were filtered using Whatman no.1 filter paper, evaporated and the crude methanolic extracts were subjected to GC-MS profiling and antibacterial activity.

Gas chromatography and mass spectroscopy (GC-MS) analysis
Gas chromatography and mass spectrometry were performed to analyze the qualitative and quantitative identification of organic compounds in the given sample. The potential biological compounds of S. khasianum leaf and root extracts were analyzed using GC-MS (Agilent: 7890-Jeol: AccuTOF GCV) system coupled with Elite 1 column. Helium gas was used as a carrier gas at 1 ml/min rate of flow, with an injector volume of 2 µl and 280 °C temperature. The oven temperature was raised from 40 to 280 °C with an isothermal for 5 min. The bioactive compounds were identified based on retention time, MS fragment ions generated and the percentage of these bioactive compounds was evaluated from the total peak area. The phytochemicals have been identified by comparing their MS spectrum patterns to the standard mass spectra available at the National Institute of Standards and Technology (NIST) Mass Spectra Database.

Antibacterial activity
The leaf and root methanolic extract of S. khasianum were tested for their antibacterial activity by agar well diffusion method. Luria Bertani (LB) medium was prepared, poured at 20 ml/petridish and allowed to solidify. 24-h-old bacterial cultures (Bacillus sphaericus, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) were spread uniformly onto solidified medium. Different concentrations (20, 40, 60 and 80 µg/ml) of S. khasianum leaf and root extracts reconstituted in DMSO (dimethyl sulfoxide 10%) and streptomycin standard (10 µg/ml) were loaded into wells and incubated at 37 °C for 24 h. The antibacterial efficacy of S. khasianum extracts were observed by measuring the diameter of inhibition zones emerging around the wells. The results of triplicate mean were taken and data was presented as mean ± SD of the respective triplicate.

Results
GC-MS profiling detected potential phytochemicals in S. khasianum leaf and root methanolic extracts by their molecular formula and retention time. Sixteen phytoconstituents were detected from leaf extract and thirtytwo compounds from root extract by GC-MS (Tables 1  and 2). The compounds identified with high concentration in leaf extract include Heptadecane 9-hexyl (43.65%) and Myoinositol hexaacetate (15.05%), whereas the highest compounds identified in root extract include Stigmasterol (23.18%) and cis-Vaccenic acid (9.07%) and presented in Figs. 1 and 2. The diversification of these phytoconstituents was recorded using sunburst graph ( Figs. 3 and 4). Table 3 shows the antibacterial activity of S. khasianum leaf and root extracts. The root methanol extract showed the highest inhibition zone at 80 µg/ ml of 16 ± 0.15 mm for B. sphaericus, 21 ± 0.18 mm for Escherichia coli, 17 ± 0.02 mm for Staphylococcus aureus and 19 ± 0.18 mm for Pseudomonas aeruginosa. Leaf extract at 80 µg/ml concentration showed 15 ± 0.14 mm for B. sphaericus, 16 ± 0.16 mm for Escherichia coli, 15 ± 0.01 mm for Staphylococcus aureus and 17 ± 0.11 mm for Pseudomonas aeruginosa.

Discussion
Accurate certification and studies of phytoconstituents are increasing periodically, as they are repositories of several potent drugs. Gas chromatography and mass spectroscopy (GC-MS) has been validated to be a significant tool for bioprospecting of plant bioactive compounds. However, diethyl phthalate and n-hexadecanoic acid were identified to be common in leaf and root extract of S. khasianum. Other organic compounds in leaf extract that are accountable for their wide use in medicinal aid include: Dodecanal, reported to possess highest antibacterial activity (Faridha Begum et al. 2016). Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-,octadecyl ester shows strong antifungal and antioxidant activities in Azadirachta and Thesium humile (Akpuaka et al. 2013;Belakhdar et al. 2015).
The remaining bioactive compounds analyzed were as follows: Diethyl phthalate, a phytoconstituent well known for its antimicrobial, antioxidant, plasticizer and estrogenic activities in Ceropegia bulbosa Roxb (Arora and Meena 2017). E-9-Tetradecenoic acid is reported to have analgesic, anti-inflammatory and antioxidant properties in Cassia angustifolia (Al-Marzoqi et al. 2016). The bioactive compound, Myristoleic acid reported in Sesame Seeds was known to prevent cancer (Bhatnagar and Gopala Krishna 2009).
The S. khasianum leaf methanolic extracts showed high antibacterial activity against P. aeruginosa in concentration-dependent manner, followed by E. coli, B. sphaericus and S. aureus (Fig. 3), whereas the root methanolic extract exhibited high antibacterial activity against E. coli, followed by P. aeruginosa, S. aureus and B. sphaericus. The result indicates that the S. khasianum root extract exhibited remarkable antibacterial property against P. aeruginosa and E. coli. Therefore, root methanolic extract of S. khasianum was considered as the most effective extract than leaf extract with regard to high anti-bacterial activity (Pavani and Shasthree 2021). This indicates that the root extract had more antibacterial compounds than leaf extract. Our results were in accordance with the reports on Momordica cymbalaria (Chaitanya and Pavani 2021). This study confirms that the S. khasianum extracts have significant antibacterial activity against tested bacteria.

Conclusions
The GC-MS analysis revealed the presence of 16 bioactive compounds in leaf methanolic extract and 32 bioactive compounds in root methanolic extract of S. khasianum based on their retention time, molecular weight, peak area and MS fragment ions generated. Heptadecane, 9-hexyl and stigmasterol were the predominant potential bioactive compounds identified in leaf and root extract. These extracts have shown high antibacterial activity against gram-positive and gram-negative bacteria. This study confirmed the presence of various biomolecules with significant biological properties, thereby confirming the medicinal claim and use of Solanum khasianum and making it a potential source of medicines.